Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells

Abstract

Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.

Fig. 1

Cell permeable cross-linkers BVSB and PDES for live cells cross-linking.

Fig. 2

In vitro cross-linking of Trx1 and its substrates. A) Mechanism of reducing PAPR by Trx1. B) Western blot analysis of Trx1 cross-linked with PAPR or the PAPR C239S mutant using BVSB. C) Tandem mass spectrum showing BVSB cross-linking between Trx1 and PAPR active sites. D) Western blot analysis of Trx1 cross-linked with PAPR or the PAPR C239S mutant using PDES. E) Tandem mass spectrum showing PDES cross-linking between Trx1 active site and PAPR active site.

Fig. 3

Cross-linking of Trx1 and its substrates in live E. coli cells. A) Constructs that co-expressing Trx1/PAPR and Trx1/Tpx complexes. B-E) Western blot analysis showing successful cross-linking of Trx1 and its substrate PAPR in live cells by BVSB (B) or PDES (D). No cross-linking band detected for Trx1 and PAPR C239S mutant after treating live cells with BVSB (C) or PDES (E).

Fig. 4

Identify direct interactome of Trx1 in live E. coli cells using chemical cross-linking. A) Constructs of Trx1 WT and Trx1* (Trx1-2KR) for live cell cross-linking. This mutant enables the two-step purification step to enrich cross-linked peptides. B) Workflow of a two-step purification to enrich cross-linked peptides for MS analysis. C) Western blot analysis of cell lysates obtained from BVSB or PDES treated E. coli cells, showing multiple endogenous proteins cross-linked to Trx1. D) Types of cross-linked peptides from BVSB or PDES cross-linked Trx1 complexes. E) KEGG analysis of Trx1 interacting proteins from this study. Comparison of identified cross-linked peptides and interacting proteins from this study with those identified using an unnatural amino acid BprY (F) [29], disulfide trapping(G) [5], and SNO (H) [30].

Fig. 5

Identify the SNO substrates of TXN1 in live HEK293T cells. A) Western blot analysis of cell lysates from BVSB or PDES treated 293T cells, showing multiple endogenous proteins cross-linked to TXN1. B) Types of cross-linking peptides from BVSB and PDES cross-linked TXN1 complexes. C) KEGG analysis of TXN1 interacting proteins from this study. D-E) Venn diagram showing the overlap of SNO substrates from this study with previously identified SNO modified sites using a global proteomics approach (D) and a chemical proteomics strategy (E).

Fig. 6

Validation of SNO modified proteins using a biotin switch assay. A) Cys73 of TXN1 was the main SNO site of TXN1 as validated by the biotin switch assay. (“Old” SNOC represents the negative control with decayed SNOC, and “new” SNOC is freshly prepared SNOC). B) Biotin switch assays confirmed SNO-modification on EEF2, CHEK1, PLK1, β-tublin, and Caspase 6. C) Tandem mass spectrum supporting a cross-link between TXN1(73)-CASP6(163). D) TXN1 catalyzed transnitrosylation on caspase 6.

Fig. 7

Identify direct interactome of TRP14 in live HEK293T cells. A) Western blot analysis of cell lysates from BVSB or PDES treated 293T cells, showing multiple endogenous proteins cross-linked to TRP14. B) Types of cross-linked peptides from BVSB and PDES cross-linked TRP14 complexes. C) Venn diagrams showing overlap of identified cross-linked peptides from this study with published SNO modified sites. D）Cross-linked peptides identified by TRP14 and TXN1 cross-linking.